DESCRIPTION: Using a cultured ovine lens system, Dr. Louis will pursue 6 specific aims: 1.Determine whether IP3, Ca2+ or cADPR (cyclic ADP-ribose) are responsible for propagation of cell-cell Ca2+ waves in the lens. Each of these will be microinjected into single cells in culture and determining whether the resulting cell-cell Ca2+ waves are affected by a variety of pharmacological reagents that are specific for different enzymes and channels used by 2nd messenger molecules. 2.Absolute concentrations of IP3 and cADPR will be measured following addition of agonists which elevate Ca2+. Cell surface receptors that mediate the agonist or mechanical stimulation-dependent elevation of Ca2+ will be determined. Then the PtdIP2-phospholipase C(s), ADP-ribosyl cyclase(s) will be identified. Then the channel proteins responsible for elevating Ca2+ identified. 3.Identify the agonists and enzymes responsible for cAMP signaling to determine the relationship between agonists that elevate Ca2+ and the activation of adenylate cyclase. Effects of membrane permeable activators of protein kinases, phosphatase inhibitors and calmodulin inhibitors will be studied to determine whether kinases or calmodulin affect Ca2+ waves. 4.The more differentiated lens "fibers" have higher resting Ca2+. Experiments will determine if the "fibers" have different Ca2+ regulatory mechanisms. 5.Effects of oxidizing agents on lens cell cytosolic Ca2+ will be determined using both membrane permeant and impermeant -SH reagents. 6.The roles of the different lens connexins in mediating the Ca2+ waves will be studied by measuring Ca2+ permeability of both homotypic and heterotypic gap junctions in HeLa cells.